1. Field
This disclosure relates generally to the preparation of human blood plasma proteins and specifically with the preparation of alpha.sub.1 -antichymotrypsin (ACT) from a mixture of ACT, alpha.sub.1 -proteinase inhibitor (PI) and antithrombin III (AT-III) such as that found in Cohn Fraction IV-1 paste suspension obtained from human plasma.
2. Prior Art
Before the Second World War, scientists and physicians discovered that human plasma could be used in blood replacement therapy. During the War, difficulties of supply and storage of whole blood and plasma meant battlefield shock could not be treated as effectively as possible. The need for plasma proteins which could be stored and used on the battlefield led Cohn and others (U.S. Pat. No. 2,390,074 (1945) and the J. Amer. Chem. Soc., 68; p459 (1946)) to discover that proteins present in plasma could be fractionated by selective precipitation in the presence of water-soluble organic solvents or neutral salts. For a review of plasma fractionation see, "The Plasma Proteins", Second Edition, Volume III, pp 548-550, Academic Press, New York, N.Y. (1977). The concentrated protein mixtures could then be introduced into patients as needed. For example, if excess bleeding was the problem, the physician could inject a fibrinogen-enriched fraction. If the patient suffered burns or other traumatic injury where the loss of plasma exceeded that of red blood cells, the physician could use albumin, the colloid-osmotic regulator of plasma.
In Cohn fractionation, ethanol is added to plasma and the pH is lowered at sub-zero temperatures to selectively precipitate protein. After the precipitate is separated from the supernatant, the pH of the supernatant is lowered, and/or more alcohol is added to precipitate another fraction.
Today, this method of fractionation is still being used to separate biologically active proteins that possess certain therapeutic qualities. For instance, Factor VIII or anti-hemophilic factor is useful against hemophilia; plasminogen, a precursor of plasmin, is used in the treatment of acute thromboembolic disorders; gamma globulins, including immune serum globulin and intravenous gamma globulin, are employed in the treatment of congenital gamma globulin deficiency, measles, poliomyelitis and hepatitis A and B; fibronectin has been identified as active in treatment of burns, shock, cancer, etc.; anti-thrombin III is a coagulation inhibitor; cryoprecipitate itself may be used directly for classic hemophilia; Plasma Protein Fraction and albumin are useful in treatment of shock due to burns, crushing injuries, abdominal emergencies, and any other trauma producing a predominant loss of plasma fluids but not red cells; and .alpha..sub.1 -proteinase inhibitor can be employed in the treatment of emphysema.
Human .alpha..sub.1 -antichymotrypsin (ACT) is a serine protease inhibitor that has, until now, only been isolated from human plasma or serum. Although the precise biological function of ACT has not yet been determined, it appears to be a multifunctional protein and its use for various therapies has been suggested. (See, for example, U.S. Pat. No. 5,008,242 to J. Lezdey, et al.)
There is evidence to indicate that an important function of ACT is the inhibition of proteases, such as chymotrypsin-like protease, mast cell chymase, leukocyte cathepsin G (see Beatty, K., Bieth, J., Travis, J.: Kinetic of association of serine proteinases with native and oxidized .alpha..sub.1 -proteinase inhibitor and .alpha..sub.1 -antichymotrypsin, J. Biol. Chem. 1980; 255:3931-3934) and pancreatic elastase (see Laine, A., Davril, M. Rabaud, M., et al.: Human serum .alpha..sub.1 -antichymotrypsin is an inhibitor of pancreatic elastases, Eur. J. Biochem. 1985; 151:327-331 and Davril, M., Laine, A., Hayem, A.: Studies on the interactions of human pancreatic elastase 2 with human .alpha..sub.1 -proteinase inhibitor and .alpha..sub.1 -antichymotrypsin, Biochem. J. 1987; 245:699-704).
The biological properties of intact, cleaved and complexed forms of ACT indicate the proteolytic-character of the protein may have a potential role for therapeutic use in regulating infectious disease, pancreatitis, lung disease and skin inflammation (see Rubin, H.: The biology and biochemistry of antichymotrypsin and its potential roles as a therapeutic agent, Biol. Chem. Hoppe-Sayler 1992; 373(7):497-502).
J. Travis et al. purified ACT to homogeneity from a human plasma pool (Travis, J., Garner, D., Bowen, J.: Human .alpha..sub.1 -antichymotrypsin: purification and properties, Biochemistry 1978; 17:5647-5651).
T. Katsunuma and his colleagues purified a DNA-binding protein, thought to be a tumor marker, to homogeneity (Katsunuma, T. et al.: Purification of a serum DNA binding protein (64DP) with a molecular weight of 64,000 and its diagnostic significance in malignant diseases, Biochem. and Biophys. Res. Comm., 93(2):552-557 (1980)). Later, the DNA-binding protein was found to be ACT. Katsunuma used human serum as the starting material. They first eluted 64DP from DEAE Sephadex with 225 mM NaCl. After dialysis, the 64DP was further purified on DNA Cellulose. Finally to achieve homogeneity, 64DP was precipitated with ammonium sulfate and separated from contaminating proteins on a size exclusion column.
In addition to isolation from whole plasma, human ACT has been cloned, sequenced and expressed in Escherichia coli (see Rubin, H. et al.: Cloning, expression, purification and biological activity of recombinant native and variant human antichymotrypsins, J. Biol. Chem. 1990; 265:1199-1207). Rubin et al. used a Sepharose Fast Q column to separate ACT activity from the crude bacterial lysate. The partially purified ACT was then adsorbed to DNA Cellulose and eluted in 350-400 mM KCl.
Because of the potential uses of ACT, there is now a need for more efficient ways of preparing large quantities of ACT from human plasma especially from plasma fractions that also include varying amounts of PI and AT-III.
Unexpectedly, we discovered that ACT can be purified from Cohn Fraction IV-1. Fraction IV-1 contains many different proteins and difficulties in separation preclude most commercial uses of this plasma fraction. Isolation of ACT would therefore be a beneficial use of this otherwise underutilized and normally discarded plasma fraction.
Compared to human serum, Cohn Fraction Paste IV-1 contains higher concentrations of proteins, such as PI and AT-III, that are closely related to ACT and would be expected to co-purify with ACT. During the fractionation process to IV-1 paste, ACT is only concentrated 1-2 fold, while PI is concentrated 3-10 fold and AT-III is concentrated 2-3 fold. The concentrations of the proteins in the IV-1 paste are 30 mg PI/g paste, 5 mg AT-III/g paste and 5-10 mg ACT/g paste. Considering the difficulty in separating ACT from these other proteins and its status as a minor component, it would be considered unlikely that Fraction IV-1 paste would be a suitable source of ACT. Surprisingly, we were able to isolate 2-3 g ACT at a purity .gtoreq.90% from approximately 2.9 kg of IV-1 paste using a modified form of the procedure of Katsunuma, et al.
In addition to its demonstrated use an as in vitro serine protease inhibitor (e.g., it can be used as a reagent for PI studies), we have proposed how ACT may be of potential therapeutic use. Details of our discoveries are disclosed below.